Contrastive Study Of Chemical Composition And Pharmacodynamic Dosage Between The Chinese Herbal Ultrafine Powdering And Traditional Processed

1. Objectives and significanceApplication of Traditional Chinese Medicine (TCM) is for several thousand years of history and to protect people's health made significantly contribution in China. However, with the scientific and technological progress, people's living standards improving, the Chinese herbal traditional processed (TP) boiling trouble and taking the inconvenience severely restricted the development of clinical medicine. In the 1990s, ultrafine grinding technology whose purpose was cytoderm broken was successfully applied to Chinese herbal processed, and by which more than 400 kinds of Chinese herbs had been made into ultrafine powdering(UP). Dosage of the Chinese herbs can be reduced because the effective chemical components was dissolved more and effect was improved after ultrafine grinding. Because of this, dosage of UP must be redetermined before clinical using. But the dose-effect relationship of UP is not clear yet. Traditional Chinese Medicine (TCM) formulae is a primary therapeutic way using Chinese medical clinic. Here we used two calssic formula Xiao Chai Hu Decoction(小柴胡汤)and Ge Gen Qin Lian Decoction(葛根芩连汤)as a example to contrast the chemical components and relativity of the pharmacodynamic dosage between UP and TP for clinical using. 2. Methods2.1 Comparitive study of chemical components between UP and TPWater-loaded slide within few UP was observed to calculate the diameter and the rate of Wall-broken cells accounted for intact cells under the microscope. The baicalin component of Xiao Chai Hu Decoction and puerarin, berberine, baicalin components of Ge Gen Qin Lian Decoction were analyzed by TLC between UP and TP. The water-soluble extracts of Xiao Chai Hu Decoction and Ge Gen Qin Lian Decoction were determined and compared between UP and TP.The chromocor content (mg·g-1) of UP was determined by ultraviolet-visible spectrophotometry (UV) under different solvent volume (5 times,10 times and 20 times) and compared with that of TP. Under the same solvent volume, it was determined by UV at different times and compared with that of TP. The baicalin content (mg·g-1) of UP was determined by HPLC under different solvent volume (5 times,10 times and 20times) and compared with that of TP. Under the same solvent volume (20 times), it was determined by HPLC at different times and compared with that of TP.2.2 Comparetive study of Pharmacodynamic dosage between UP and TPIt was tested for organ index including liver, spleen, thymus and serum transaminase activity and liver pathological change between UP and TP in immunological liver injury model induced by Bacille calmette-guerin (BCG) in mice. The effect of UP and TP on proliferation of mouse spleen lymphocytes was analyzed by MTT. It was observed for the effect on carbon powder propulsion of intestinal in mice and on bacteriostasis in Vitro between UP and TP in order to understand the relationship of pharmacodynamic dosage.3. Results3.1 Comparitive study of the chemical components between UP and TP3.1.1 Diameter determination of UPIt was observed that Wall-broken cells of UP accounted for more than 95% of vision field, which diameter was generally from 2μm to 30μm. Intact cells were not be seen except rare sclereid and wood fiber under the microscope.3.1.2 Thin-layer chromatography (TLC) between UP and TPThe spots of UP and TP were showed at the same location as the same as reference standard, while negative reference was not. And the spot of UP was clearer than that of TP. It proved that chemical component of the Chinese herb was not changed and component of pharmacodynamics was reserved after ultrafine grinding.3.1.3 Comparitive content of the water-soluble extract between UP and TPWater-soluble extract content of UP was Significantly dissolved largely (P<0.01), about 1.5～2 times as much as that of TP under the same conditions. It proved that water-soluble components dissolved were increased after ultrafine grinding.3.1.4 Comparitive content of chromocor and baicalin component between UP and TP of Xiao Chai Hu DecoctionUnder the same amount of solvent conditions, chromocor and baicalin content of UP were Significantly dissolved largely (P<0.01), about 1.5-2 times as much as that of TP. The content of active ingredients were increased with the increase of the solvent volume. Maximum content of the active ingredients were dissolved under 20 times amount of solvent conditions.3.1.5 Comparison on chromocor and baicalin dissolution between UP and TP of Xiao Chai Hu DecoctionExperimental results showed that immersion (or boiling) different times, chromocor and baicalin content of UP were significantly more than that of TP, in comparison with statistical had significance (P<0.05); while groups of UP compared, it was no significant difference (P>0.7). It proved that the active ingredients were dissolved quickly in a short time. Chromocor and baicalin content of TP was positively related to the boiling time.3.1.6 Comparitive content of berberine alkaloids and puerarin between UP and TP of Ge Gen Qin Lian DecoctionExperimental results showed that the content of berberine alkaloids and puerarin was separately 1.74 times and 1.37 times as much as that of TP under the same solvent volume conditions. It provided that the active ingredients dissolved were increased after ultrafine grinding.3.2 Comparitive study of Pharmacodynamic dosage between UP and TP3.2.1 Comparitive study of Pharmacodynamic dosage between UP and TP of Xiao Chai Hu DecoctionIn immunological liver injury experiment in mice, compared with the control group, serum ALT and AST activity of model group were significantly increased (P <0.05). Serum ALT activity was significantly reduced in bifendate group,UPl group, UP2 group,TP1 group and TP2 group in the immunological liver injury in mice (P <0.05); So were serum AST activity in UP1 group, UP2 group and TP1 group. Serum AST activity was not significantly reduced by bifendate. The experimental data analysis shows that the lowest effective dosage of UP on serum transaminase was 1/4 times as much as that of TP. Compared with the model group, the degree of liver pathological damage can be significantly mitigated in bifendate group, UP1 group and TP1 group (P<0.05). It was proved that Xiao Chai Hu Decoction has a protective effect on liver injury. The lowest effective dosage of UP on liver pathology was 1/2 times as much as that of TP. Compared with the control group, spleen index of model group had statistically significant difference (P<0.05), while liver and thymus index has no difference. Experimental results showed that liver, spleen, thymus index had not significantly recovered in drug-treated groups.Compared with the model group, UP1 group, UP2 group, TP1 group and TP2 group could significantly inhibit spleen lymphocytes proliferation in the experiment (P<0.05). Effect of bifendate group on spleen lymphocytes proliferation was not significant. The lowest effective dosage of UP on spleen lymphocytes proliferation was 1/2 times lower than that of TP.3.2.2 Comparitive study of Pharmacodynamic dosage between UP and TP of Ge Gen Qin Lian DecoctionCompared with the control group, berberine group,UP1～4 group and TP1 group could significantly inhibit the small intestinal propulsion in mice (P<0.05). The data indicated that the lowest effective dosage of UP was 1/16 times lower than that of TP on inhibiting the small intestinal propulsion. In antibacterial experiment, compared with the control group, amoxicillin group,UP1 group and TP1 group could significantly inhibit Staphylococcus aureus, Shigella flexneri and Shigella dysenteriae growth(P<0.05). The experimental results showed that bacteriostasis of UP was better than that of TP in the same doses, and minimal inhibitory dosage of UP was 1/2 times lower than that of TP.4. Conclusion4.1 It was observed that Wall-broken cells of UP were accounted for more than 95% of the intact cells, almost granular material, which diameter was generally from 2μm to 30μm. Intact cells were not be seen except rare sclereid and wood fiber under the microscope4.2 Chemical composition of pharmacodynamics was reserved as the same as TP because atomic or molecular structure was not changed after ultrafine grinding. The spots of UP and TP were showed at the same location as the same as reference standard, it proved that chemical component of the Chinese herb was not changed and component of pharmacodynamics was reserved after ultrafine grinding.4.3 Active components of UP were dissolved largely and quickly compared with that of TP. The main active ingredients of Chinese herb were usually reserved in the cells. So the active ingredients were dissolved directly and largely after ultrafine grinding. The medical component content of UP was 2 times more than that of TP. Without boiling, active components of UP were dissolved largely and quickly in 10 minutes after boiling water soaking.4.4 Dosage of UP could be reduced in clinic. Pharmacodynamics experimental results showed that different formulas, different observation choiced may affect the result of the dose-effect relationship between TP and UP. In protection immunological liver injury in mice of Xiao Chai Hu Decoction experiments, ALT activity level could be significantly reduced by UP, which dosage was 1/4 time as much as that of TP, so were AST and liver pathology by UP which dosage was 1/2 time as much as that of TP. The small intestinal propulsion was inhibited by UP, which dosage was 1/16 time as much as that of TP, but the effects on antibacterial in Vitro was significant by UP which dosage was 1/2 time as much as that of TP.